关键词： IQGAP1   reactive oxygen species   vascular endothelial growth factor   endothelial cell   cell migration  

摘要： Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.

关键词： cell motility   lammelipodium   Cdc42   Rac1   calmodulin  

摘要： IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 muM K-d) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K-d of similar to47 muM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. (C) 2004 Wiley-Liss, Inc.

关键词： cell-free assay   E-cadherin   endocytosis   Rho family small G proteins   IQGAP1  

摘要： E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.

关键词： Biochemical engineering  

摘要： Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to similar to 2.5 times the normal shear stress, or similar to 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation ( cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca2+](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

关键词： calmodulin   IQGAP1   calcium   F-actin   Cdc42   Rac1  

摘要： IQGAPI regulates cytoskeletal dynamics through interactions with the Rho family GTPases Racl and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ mwotifs of IQGAPI negatively influences these IQGAPI interactions. Although, calmodulin interacts with IQGAPI in the absence of Ca2+ and Was suggested to exhibit reduced binding when Ca2(+) bound, recent reports show substantially greater binding when Ca2+ is present. Binding evaluations have primarily relied on IQGAPI interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca2+-dependence of calmodulin interaction with native IQGAPl using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAPI was Ca2+-independent. being between 5- and 20-fold greater in the absence than in the presence of Ca2+ lit addition, calmodulin in- teraction with IQGAPl was negatively regulated by buffer [Ca2-] (IC50 = 3.4 x 10(-7) M). Regulation was specific to Ca2+, as Ba2+ was similar to400-fold less effective than Ca2+ at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAPI and that the N- and C-terminal pair of EF hands are important for Ca2- sensitivity. These data indicate that calmodulin may disassemble from IQGAPI to facilitate IQGAPI interaction with effectors of cytoskeletal reorQanization durina conditions of cell activation that prornote increased cytosolic [Ca2+] (C) 2004 Elsevier Inc. All rights reserved.

关键词： Rho family   IQGAP1   APC  

关键词： IQGAP1   actin   E-cadherin